Technical Report | Published:

A rapid and efficient single-cell manipulation method for screening antigen-specific antibody–secreting cells from human peripheral blood

Nature Medicine 15, 1088–1092 (2009) | Download Citation

Subjects

Abstract

Antigen-specific human monoclonal antibodies (mAbs) are key candidates for therapeutic agents. However, the availability of a suitable screening system for antigen-specific antibody–secreting cells (ASCs) is limited in humans. Here we present a unique method for detecting individual ASCs using microwell array chips, which enables the analysis of live cells on a single-cell basis and offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for identifying and recovering objective ASCs. We applied the system to detect and retrieve ASCs for hepatitis B virus and influenza viruses from human peripheral blood lymphocytes and produced human mAbs with virus-neutralizing activities within a week. Furthermore, we show that the system is useful for detecting ASCs for multiple antigens as well as for selection of ASCs secreting high-affinity antibodies on a chip. Our method can open the way for the generation of therapeutic antibodies for individual patients.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

Rent or Buy

All prices are NET prices.

References

  1. 1.

    , , & Of mice and men: hybridoma and recombinant antibodies. Immunol. Today 21, 364–370 (2000).

  2. 2.

    & Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15, 553–557 (1997).

  3. 3.

    , , & Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552–554 (1990).

  4. 4.

    , , & Making antibodies by phage display technology. Annu. Rev. Immunol. 12, 433–455 (1994).

  5. 5.

    & Antibody production of a human EBV-transformed B cell line and its heterohybridoma and trioma cell line descendants in different culture systems. Hum. Antibodies Hybridomas 5, 98–104 (1994).

  6. 6.

    & Requirements for the establishment of high-titered human monoclonal antibodies against tetanus toxoid using the Epstein-Barr virus technique. J. Immunol. 127, 1275–1280 (1981).

  7. 7.

    et al. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat. Med. 10, 871–875 (2004).

  8. 8.

    & Man-made antibodies. Nature 349, 293–299 (1991).

  9. 9.

    , & Single, antigen-specific B cells used to generate Fab fragments using CD40-mediated amplification or direct PCR cloning. Biotechniques 18, 862–869 (1995).

  10. 10.

    , , , & A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. Proc. Natl. Acad. Sci. USA 93, 7843–7848 (1996).

  11. 11.

    et al. Isolation of human antibody repertoires with preservation of the natural heavy and light chain pairing. J. Mol. Biol. 358, 764–772 (2006).

  12. 12.

    et al. MAC-CCD system: A novel lymphocyte microwell-array chip system equipped with CCD scanner to generate human monoclonal antibodies against influenza virus. Lab Chip 9, 158–163 (2009).

  13. 13.

    et al. Cell-microarray analysis of antigen-specific B-cells: single cell analysis of antigen receptor expression and specificity. Cytometry A 71, 961–967 (2007).

  14. 14.

    et al. Single lymphocyte analysis with a microwell array chip. Cytometry? 71, 1003–1010 (2007).

  15. 15.

    et al. Single-cell microarray for analyzing cellular response. Anal. Chem. 77, 8050–8056 (2005).

  16. 16.

    & Structure and mechanism of the M2 proton channel of influenza A virus. Nature 451, 591–595 (2008).

  17. 17.

    et al. Structural basis for the function and inhibition of an influenza virus proton channel. Nature 451, 596–599 (2008).

  18. 18.

    et al. Rapid cloning of high-affinity human monoclonal antibodies against influenza virus. Nature 453, 667–671 (2008).

  19. 19.

    , & Amplification and analysis of cDNA generated from a single cell by 5′-RACE: application to isolation of antibody heavy and light chain variable gene sequences from single B cells. Biotechniques 40, 469–470, 472, 474 passim (2006).

  20. 20.

    , , , & A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nat. Biotechnol. 24, 703–707 (2006).

  21. 21.

    et al. Comparison of the influenza virus-specific effector and memory B-cell responses to immunization of children and adults with live attenuated or inactivated influenza virus vaccines. J. Virol. 81, 215–228 (2007).

  22. 22.

    & Method of anisotropically etching silicon. US patent 5,501,893 (1996).

  23. 23.

    et al. Infection of a human hepatoma cell line by hepatitis B virus. Proc. Natl. Acad. Sci. USA 99, 15655–15660 (2002).

  24. 24.

    et al. Rapid focus reduction neutralization test of influenza A and B viruses in microtiter system. J. Clin. Microbiol. 28, 1308–1313 (1990).

Download references

Acknowledgements

We thank S. Hirota for technical assistance and K. Hata for secretarial work. We are very grateful to M. Isobe and N. Kurosawa, Laboratory of Molecular and Cellular Biology, Graduate School of Science and Engineering, University of Toyama, for their generous supply of an antibody expression system. We thank H. von Boehmer, Dana-Farber Cancer Institute, Harvard Medical School, for 110TC cells. This research was supported by grants from the Toyama Medical Bio-Cluster Project and a Grant-in-Aid of the Ministry of Education, Culture, Sports, Science and Technology, Japan. A.J., K.K. and S.Kadowaki were supported by a postdoctoral fellowship from the Toyama Medical Bio-Cluster Project.

Author information

    • Aishun Jin
    • , Tatsuhiko Ozawa
    •  & Kazuto Tajiri

    These authors contributed equally to this work.

Affiliations

  1. Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.

    • Aishun Jin
    • , Tatsuhiko Ozawa
    • , Kazuto Tajiri
    • , Sachiko Kondo
    • , Koshi Kinoshita
    • , Shinichi Kadowaki
    • , Hiroyuki Kishi
    •  & Atsushi Muraguchi

  2. SC World Inc., Toyama, Japan.

    • Aishun Jin

  3. The Third Department of Internal Medicine, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.

    • Kazuto Tajiri
    •  & Toshiro Sugiyama

  4. Central Research Institute, Toyama Industrial Technology Center, Takaoka, Toyama, Japan.

    • Tsutomu Obata

  5. Department of Infectious Diseases, Osaka Prefectural Institute of Public Health, Osaka, Japan.

    • Kazuo Takahashi

Authors

  1. Search for Aishun Jin in:

  2. Search for Tatsuhiko Ozawa in:

  3. Search for Kazuto Tajiri in:

  4. Search for Tsutomu Obata in:

  5. Search for Sachiko Kondo in:

  6. Search for Koshi Kinoshita in:

  7. Search for Shinichi Kadowaki in:

  8. Search for Kazuo Takahashi in:

  9. Search for Toshiro Sugiyama in:

  10. Search for Hiroyuki Kishi in:

  11. Search for Atsushi Muraguchi in:

Contributions

A.J. established the ISAAC protocol; T. Ozawa analyzed influenza-specific antibodies; K. Tajiri analyzed HBs antigen–specific antibodies; T. Obata developed microwell array chip for ISAAC; S. Kondo and K.K. constructed antibody expression vectors; S. Kadowaki analyzed cells; K. Takahashi analyzed influenza virus neutralization activity; T.S. supervised the project; and H.K. and A.M. organized and conducted the project.

Competing interests

The immunospot array assay on a chip (ISAAC) procedure has been registered for Japan Patent Office patent JP 4148367.

Corresponding author

Correspondence to Hiroyuki Kishi.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figs. 1–3, Supplementary Table 1 and Supplementary Methods

To obtain permission to re-use content from this article visit RightsLink.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nm.1966